Figure 2.
RNF12/RLIM E3 Ubiquitin Ligase Is Selectively Phosphorylated by SRPKs at a SR-Rich Motif
(A) RNF12-deficient (Rlim−/y) mESCs were transfected with WT RNF12 or the indicated point mutants and RNF12 SR-motif phosphorylation analyzed by phos-tag immunoblotting for RNF12. Fully phosphorylated (4-P) and unphosphorylated (0-P) RNF12 SR-motifs are indicated by open (○) and closed (●) circles, respectively. RNF12 4xSA = S212A/S214A/S227A/S229A.
(B) Rlim−/y mESCs were transfected with the indicated RNF12 constructs and lysates treated with λ-phosphatase and analyzed by phos-tag immunoblotting for RNF12. Unphosphorylated recombinant RNF12 is included as a control.
(C) mESCs were treated with 10 μM of the following kinase inhibitors: AZ-191 (DYRK1B), KH-CB19 (CLK-DYRK), CLK-IN-T3 (CLK), SPHINX31 (SRPK1), SRPKIN-1 (pan-SRPK), CHIR-99021 (GSK-3), PD-0325901 (MEK1/2), VX-745 (p38), JNK-IN-8 (JNK), RO-3306 (CDK1), and flavopiridol (CDK7/9) for 4 h and RNF12 SR-motif phosphorylation analyzed by phos-tag immunoblotting for RNF12. RNF12 4xSA is included as an unphosphorylated control.
(D) mESCs were treated with the indicated concentrations of SRPKIN-1 for 4 h and RNF12 SR-motif phosphorylation analyzed by phos-tag immunoblotting for RNF12.
(E) mESCs were treated with 10 μM SRPKIN-1 for 4 h and RNF12 phosphorylation analyzed from HA-RNF12 immunoprecipitates via RNF12 phos-tag and phospho-Ser214 immunoblotting using multiplex infrared immunoblot.
(F) Phosphorylated peptides detected by mass spectrometry following in vitro phosphorylation of RNF12 by SRPK1. pS, phospho-serine.
(G) Autoradiography of RNF12 WT or S212A/S214A/S227A/S229A (4xSA) following a radioactive kinase reaction with SRPK1, SRPK2, or SRPK3. RNF12 protein is detected by Coomassie staining.
(H) Srpk1+/+ and Srpk1−/− mESCs were transfected with control or SRPK2 siRNA and RNF12 SR-motif phosphorylation analyzed by phos-tag immunoblotting for RNF12. SRPK2, SRPK1, RNF12, and ERK1/2 levels were determined by immunoblotting.